On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis

Torre, Françoise de la; Bennardo, Thérèse; Šebo, Peter; Szulmajster, Jekisiel

doi:10.1016/0006-291x(89)91828-7

Cited by 19 publications

(10 citation statements)

References 15 publications

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“…Altogether these results, added to those previously reported (de la Torre et al 1989), lead to the assumption that the role of the 51-kDa protein is not only to participate in a proteolytic activation of the 42-kDa protein but may, in addition, be involved at the post-translational level in a covalent modification of the 41-kDa protein required for expression of its activity.…”

Section: Discussionsupporting

confidence: 57%

“…As in the case of the wild-type toxin (de la Torre et al 1989), the activity of the truncated toxin is restored when the 51.4-kDa protein is added prior to testing (not shown). Thus, the truncated toxin still requires the presence of the synergistic 51 .CkDa protein to exhibit larvicidal activity.…”

Section: Biological Activity Of Toxins With C-and N-terminal Deletionsmentioning

confidence: 73%

“…Their nucleotide sequences revealed the presence of two genes coding for 51.4-kDa and 41.9-kDa proteins (Arapinis et al 1988;Hindley and Berry 1987;. These genes were expressed in the heterologous hosts Escherichia coli, Bacillus subtilis and Anacystis nidulans, conferring on them toxicity for mosquito larvae (Tandeau de Marsac et al 1987;de la Torre et al 1989). …”

mentioning

confidence: 99%

“…Following purification, toxicity was found to be associated with the 41.9-kDa protein (Baumann et al 1985;Sgarella and Szulmajster 1987). Upon expression of the toxin genes in Escherichia coli, the recombinant-produced toxin is active only when the 51.4-kDa and 41.9-kDa proteins are either co-expressed in the same cell or produced in separate cultures and mixed prior to testing de la Torre et al 1989). In B. subtilis sporulating cells, however, the toxin activity is expressed in the absence of the 51.4-kDa protein which indicates that the activation of the 41.9-kDa protein can be accomplished by host enzyme(s).…”

mentioning

confidence: 99%

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Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity

Šebo

Bennardo

Torre

et al. 1990

European Journal of Biochemistry

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The two genes of Bacillus sphaericus 1953M coding for the 51.4-kDa and 41.9-kDa proteins are both required for the expression of the active larvicidal toxin in Escherichia coli. The minimal size of the active peptide of the 41.9-kDa toxin was defined by in vitro deletion analysis of the gene and found to consist of 338 amino acids (38.3 kDa). N-terminal deletions past the Ile18 residue and C-terminal deletions past the His352 residue result in the loss of toxic activity and rapid degradation of such modified toxins by host proteases. The minimal active 38.3-kDa peptide produced in E. coli seems to mimick the stable processed form of the toxin found in larval midguts. However, it still requires the action of the synergistic 51.4-kDa protein for the larvicidal activity.

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Section: Discussionsupporting

confidence: 57%

Section: Biological Activity Of Toxins With C-and N-terminal Deletionsmentioning

confidence: 73%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity

Šebo

Bennardo

Torre

et al. 1990

European Journal of Biochemistry

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“…These crystals contain two major polypeptides of 41.9 kDa and 51.4 kDa, as deduced from DNA sequence data (Hindley and Berry, 1987;Baumann et al, 1988;Arapinis et al, 1988; for complete review see Baumann et al, 1991). Bacillus subtilis or Escherichia coli expressing either of the genes encoding these two proteins are poorly toxic or even non-toxic for mosquito larvae (Baumann et al, 1988;de la Torre et al, 1989;Broadwell et al, 1990). However, transformants expressing both genes are fully toxic, indicating that the protein crystal of B. sphaericus acts as a binary toxin (Broadwell et al, 1990;Baumann and Baumann, 1991).…”

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confidence: 99%

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush‐border membranes from mosquito larvae

Nielsen-LeRoux

Charles

1992

European Journal of Biochemistry

124

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The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from C u k x pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Bacillus sphaericus is a Gram-positive spore-forming bacterium and is one of the most promising agents for the control of mosquitoes. Some strains produce a very high larvicidal activity, especially for mosquito species belonging to the Culex and Anopheles genera, while they are almost inactive on Aedes species. The insecticidal activity is directly related to the synthesis of parasporal protein crystals during sporulation (Davidson, 1983;Payne and Davidson, 1984;Kalfon et al., 1984;Baumann et al., 1985). These crystals contain two major polypeptides of 41.9 kDa and 51.4 kDa, as deduced from DNA sequence data (Hindley and Berry, 1987;Baumann et al., 1988;Arapinis et al., 1988; for complete review see Baumann et al., 1991). Bacillus subtilis or Escherichia coli expressing either of the genes encoding these two proteins are poorly toxic or even non-toxic for mosquito larvae (Baumann et al., 1988;de la Torre et al., 1989;Broadwell et al., 1990). However, transformants expressing both genes are fully toxic, indicating that the protein crystal of B. sphaericus acts as a binary toxin (Broadwell et al., 1990;Baumann and Baumann, 1991).Little is known about the mode of action of these toxins, except that they appear to exert cytopathological effects (Davidson, 1981;Karch and Coz, 1983; Charles, 1987) by binding to susceptible mosquito larval midgut cells (Davidson et al., 1987;Davidson, 1988Davidson, ,1989. In addition, both proteins bind to the gastric caecae and posterior midgut of Culex larvae when administered together (Davidson et al., 1990).In this report, we demonstrate that B. sphaericus binary toxin specifically binds to susceptible mosquito larval midgut membranes, and we measure the affinity of the toxin for its putative membrane receptor. MATERIALS AND METHODS Insects and toxicity assaysLarvae of Culexpipiens ssp. pipiens L. (Montpellier strain) and Aedes aegypti L. (Bora-Bora strain) were reared on cat biscuits at 28 "C. Adult populations were maintained on honey solution, at 2 8 T , in 80% relative humidity with a 16 hi8 h photoperiod. Females were fed on guinea pigs.Toxicity assays were performed on early fourth-instar larvae, in 5-cm diameter Petri dishes containing 10 ml bacterial or crystal suspension, or soluble protein dilutions in demineralized water. Dead larvae were counted after 24 h and 48 h, and lethal concentrations killing 50% of the population (LC,,) evaluated by probit analysis (Finney, 1971). Brush-border membrane isolationBrush-border membrane fractions (BBMF) were prepared from midguts of C. pipiens and A . aegypti larvae following methods modified from those of Biber et al. (1981; described in Wolfersberger et al., 1987) and Houk et al. (1986). Fourthi...

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Regulation of mosquitocidal toxin synthesis in Bacillus sphaericus

Ahmed

Mitchell

Priest

1995

Appl Microbiol Biotechnol

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Translational lacZ fusions to the promoters of the parasporal, crystal protein (binary toxin) and 100-kDa ADP-ribosylating mosquitocidal toxin genes of Bacillus sphaericus were prepared and expression of the toxin genes monitored as beta-galactosidase activity. Transcription of the crystal protein gene fusion began immediately before the end of exponential growth and continued into stationary phase in both B. sphaericus and Bacillus subtilis but accompanied exponential-phase growth in Escherichia coli. Expression of this fusion was severely delayed in a B. subtilis spo0A mutant and decreased relative to the wild type in a B. subtilis spoIIAC background. beta-Galactosidase activity from the 100-kDa toxin gene fusion was restricted to early exponential phase in B. sphaericus, but in B.subtilis it continued into late exponential phase. Expression was about eightfold lower in B. sphaericus than B. subtilis suggesting an element of negative control in the native host.

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On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis

Cited by 19 publications

References 15 publications

Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity

Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush‐border membranes from mosquito larvae

Regulation of mosquitocidal toxin synthesis in Bacillus sphaericus

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